In addition to upregulating TNF production (J Immunol 1994; Blood 1997), NO was found to modulate IL-8 mRNA levels and IL-8 production in human neutrophil preparations (J Infect Dis, 1998). Investigation of TNF regulation by NO resulted in the description of a cGMP-independent cAMP/SP1 signaling pathway (J Biol Chem 1997; J Biol Chem, 1999; J Biol Chem, 2003). For TNF, an upstream AP1 site coupled to the Sp1 binding sequence serves as a molecular switch, the presence of which reverses the polarity of NO responses. Mutation of the AP1 site in the proximal TNF promoter converts the NO effect from up to down-regulation. This latter promoter configuration is responsible for NO suppression of eNOS expression. The IL-8 promoter lacks a canonical Sp1 site. Unlike TNF, IL-8 regulation by NO is both cGMP and cAMP-independent. NO activation of p38 MAPK was shown to stabilize IL-8 mRNA via altered protein binding to AU-rich elements in its mRNA 3UTR (J Leuk Biol, 2004). An oligonucleotide microarray analysis in differentiated U937 cells identified more than 100 additional NO regulated genes (BMC Genomics, 2005). NO coordinated a highly integrated program of cell cycle arrest through p38 MAPK stabilization of p21 mRNA, a master regulator of the cell cycle ((J Biol Chem, 2007). Next, transcript stabilization by NO was investigated in human THP-1 cells using microarrays (Nucleic Acids Research, 2006). NO stabilization of mRNA, but suppression of translation was associated with CU-rich elements (CURE) in target transcripts and mediated by activation of ERK1/2 and the binding of hnRNP proteins to mRNA. Both NO and peroxisome proliferator-activated receptors (PPARs) protect the endothelium and regulate its function. In a crosstalk signaling pathway, PPARgamma was activated by NO through a p38 MAPK dependent signal transduction pathway (FASEB J, 2007). This mechanism may contribute to the anti-inflammatory and cytoprotective effects of NO in the vasculature. While NO up-regulated IL-1beta and TNFalpha, CO was found to decrease the expression of both. Using microarrays, early-immediate transcripts were induced by LPS and suppressed by CO (PLoS One 2009). CO blocked proximal events in NF-kappaB signal transduction, broadly suppressing inflammation. In acute respiratory distress syndrome (ARDS), combining clinically tolerated, low doses of inhaled NO and CO may have therapeutic advantages over either gas alone. As noted in our previous work, NO activates MAPK pathways and enhances inflammatory responses, effects that might counteract the toxicity and immune suppression of anthrax lethal toxin (LeTx). Furthermore, NO has been shown to interact with and alter the activity of other zinc-containing proteins. The ability of NO to inactivate LF through tryrosine nitrosylation was demonstrated in vitro. In rats, 3-morpholinosydnoime (SIN-1), a NO and superoxide donor, was shown to inactivate LeTx (American Thoracic Society, abstracts 2012 and 2014 ). Preliminary experiments have indicated that CO may activate stress kinase pathways through a G-protein coupled receptor signaling pathway. Experiments are planned to further test this hypothesis. Work demonstrating that NO/p38 MAPK activates PPARgamma was extended using PPRE reporter genes, NOS2 induction and p38 MAPK dominant negative mutant expression. Both NO and optimal PPARgamma ligand activation of PPARgamma signaling were associated with p38 MAPK phosphorylation. The role of p38 MAPK in ligand/agonist activation of PPARgamma was also shown in endothelial cells. GPR40 and PPAR&#947;, linked by p38 MAPK and PGC1&#945;, were found to function as an integrated two-receptor signal transduction pathway, a finding with implications for rational drug development (submitted 2014).